The invention relates to plasmids derived from pBluescript KS(+), comprising more than 1 SK primer sequence element, preferably 2, 7, 14, 21 and 27 repetitive SK primer sequence elements, and their use as molecular-biological markers in analytical electron microscopy.
Electron spectroscopic imaging (ESI) is a method of analytical electron microscopy (EM), which pictures the distribution of certain chemical elements within an analyzed preparation. In order to elucidate the structural organizations of biological systems, it must be possible to optically differentiate the individual macromolecular components. At present, charging with, gold particles or other particles, which are visible in refraction contrast, is used to label macromolecules for electron microscopy.
Multiple labeling experiments have been carried out in electron microscopy, where particles of various sizes are used, which enable differentiation of different target structures in a single preparation. For example, one type of molecule is linked to gold particles having a size of 5 nm while another type is linked to 10-20 nm sized particles in a double labeling experiment, to ensure that in a subsequent evaluation different molecules can be clearly localized and distinguished from one another. Large gold particles (larger than 10 nm) are disadvantageous, because of lower penetration capacity into the tissue and reduced coupling efficiency to a target molecule (Giberson, T. R., and Demaree, R. S.: The influence of immunogold particle size on labeling density. Microscopy Research and Technique, 27, 355-357, 1994). In addition, such a large particle is no longer assigned to a binding site of a target structure, because, resolution capability would be lost. If a triple labeling experiment is aimed at, the above drawbacks would become particularly striking. Only ferritin molecules, i.e. large protein units, which contain hundreds of iron atoms in their centers and can be linked to target structures, which are alternative to the gold particles. However, their electron density and detectability under the transmission electron microscope is very poor. Therefore, their use has been proved feasible only in rare cases.
On the other hand, florescence methods enable triple and quadruple labeling without any major problem in optical microscopy. Electron microscopy with the existing labeling techniques could not compete with optical microscopy. Therefore, scientists have been using optical microscopes with comparatively poor resolution capability. The development of an alternative labeling technique for gold labeling would render electron microscopy more attractive, because its advantageous labeling provides a resolution capability over 100 times as good as that of the optical microscopy. The gold labeling method for conventional transmission electron microscopy is based on the electron density of heavy metal gold and there is a demand for an alternative labeling method for ESI. This technique, which utilizes interactions between beam electrons and the atoms in the preparation, differs from conventional transmission electron microscopy. In principle, all of the elements can be detected specifically, which raises the number of elements in consideration for labeling methods. However, to establish alternative labeling methods, it is decisive to check detection limits for the elements in consideration. This means, information is required on the number of detectable element atoms per nm2 area. Therefore, the detection limits of the ESI technique are relevant. Only a few study-reports and vague indications on this parameter are available. Although the ESI technique is often used, no data on detection limits have been published to date.
There is a demand for alternative labeling methods for electron microscopy. It should be possible to readily test and assess the detectability of a marker complex.
Therefore, an object of the present invention is to provide a method of obtaining data, to evaluate the prospects of the intended experiment with the element in question and/or the marker structure in question, before time-consuming cytobiological and molecular-biological experiments are carried out. Furthermore, the parameter for the detectable number of elementary atoms per unit area shall become measurable to obtain necessary information to establish EM labeling methods.
This object is achieved by the subject matters defined in the claims section.
Until now, there has been no accurate limiting values of detectability known for the ESI detection of the various chemical elements. This is inter alia due to the fact that preparing a suitable test sample is not a trivial matter. Such a sample must have special properties. There must be regions in which the target element is available in a clearly defined amount. It must be possible to clearly detect these regions. The target element may not occur in the remaining regions. This problem has been reported by investigators who tried to record the resolution and detectability by means of grainy precipitates, using uranium, for example (see, Golla and Kohl, Micron, 28:(5), 397-406, 1997).
In one aspect, the invention provides plasmids derived from pBluescript KS(+), comprising more than 1 SK primer sequence element, preferably 2, 7, 14, 21 and 27 repetitive SK primer sequence elements.
In another aspect, the invention provides methods of analytical electron microscopy, comprising: providing the plasmid as described herein; adding a detectable element to the SK primer, thereby forming a marker complex; and imaging the marker complex by electron microscopy.
In yet another aspect, the invention provides a test kit for use in electron microscopy comprising: host E. coli JM110 bacterial cells suitable for replicating the plasmid as described herein; and a single-stranded plasmid comprising 2, 7, 14, 21, and 27 repetitive SK primer sequence elements.